Phenoxyethyl-thiourea-pyridine compounds and their use for treatment of HIV-infections

ABSTRACT

The invention provides compounds which inhibit reverse transcriptase (RT) and which inhibit replication of a retrovirus, such as human immunodeficiency virus-1 (HIV-1). The compound of the invention are phenoxyethyl-thiourea-pyridines. The invention additionally provides a method for inhibiting reverse transcriptase activity of a retrovirus, such as HIV-1, comprising contacting the retrovirus with a compound of the invention. The invention additionally provides a method for inhibiting replication of a retrovirus, such as HIV-1, comprising contacting the retrovirus with a compound of the invention.

FIELD OF THE INVENTION

[0001] The invention relates to compounds as non-nucleoside inhibitorsof reverse transcriptase that are effective against HIV, includingmutant strains of HIV, and effective in the treatment of multi-drugresistant HIV infection.

BACKGROUND OF THE INVENTION

[0002] Agents currently used to treat HIV infection attempt to blockreplication of the HIV virus by blocking HIV reverse transcriptase or byblocking HIV protease. Three categories of anti-retroviral agents inclinical use are nucleoside analogs (such as AZT), protease inhibitors(such as nelfinavir), and the recently introduced non-nucleoside reversetranscriptase inhibitors (NNI), such as nevirapine.

[0003] The recent development of potent combination anti-retroviralregimens has significantly improved prognosis for persons with HIV andAIDS. Combination therapies may be a significant factor in the dramaticdecrease in deaths from AIDS (a decrease in death rate as well asabsolute number). The most commonly used combinations include twonucleoside analogs with or without a protease inhibitor.

[0004] Nevirapine is currently the only NNI compound which has been usedin combination with AZT and/or protease inhibitors for the treatment ofHIV. A new series of effective drug cocktails will most likely involveother NNIs in combination with nucleoside and protease inhibitors as atriple action treatment to combat the growing problem of drug resistanceencountered in single drug treatment strategies.

[0005] The high replication rate of the virus unfortunately leads togenetic variants (mutants), especially when selective pressure isintroduced in the form of drug treatment. These mutants are resistant tothe anti-viral agents previously administered to the patient. Switchingagents or using combination therapies may decrease or delay resistance,but because viral replication is not completely suppressed in singledrug treatment or even with a two drug combination, drug-resistant viralstrains ultimately emerge. Triple drug combinations employing one (ortwo) nucleoside analogs and two (or one) NNI targeting RT provide a verypromising therapy to overcome the drug resistance problem. RT mutantstrains resistant to such a triple action drug combination would mostlikely not be able to function.

[0006] Dozens of mutant strains have been characterized as resistant toNNI compounds, including L1001, K103N, V106A, E138K, Y181C and Y188H. Inparticular, the Y181C and K103N mutants may be the most difficult totreat, because they are resistant to most of the NNI compounds that havebeen examined.

[0007] Recently, a proposed strategy using a knockout concentration ofNNI demonstrated very promising results. The key idea in this strategyis to administer a high concentration of NNI in the very beginningstages of treatment to reduce the virus to undetectable levels in orderto prevent the emergence of drug-resistant strains. The ideal NNIcompound for optimal use in this strategy and in a triple actioncombination must meet three criteria:

[0008] 1) very low cytotoxicity so it can be applied in high doses;

[0009] 2) very high potency so it can completely shut down viralreplication machinery before the virus has time to develop resistantmutant strains; and

[0010] 3) robust anti-viral activity against current clinically observeddrug resistant mutant strains.

[0011] Novel NNI designs able to reduce RT inhibition to subnanomolarconcentrations with improved robustness against the most commonlyobserved mutants and preferably able to inhibit the most troublesomemutants are urgently needed. New antiviral drugs will ideally have thefollowing desired characteristics: (1) potent inhibition of RT; (2)minimum cytotoxicity; and (3) improved ability to inhibit known,drug-resistant strains of HIV. Currently, few anti-HIV agents possessall of these desired properties.

[0012] Two non-nucleoside inhibitors (NNI) of HIV RT that have beenapproved by the U.S. Food and Drug Administration for licensing and salein the United States are nevirapine (dipyridodiazepinone derivative) anddelavirdine (bis(heteroaryl)piperazine (BHAP) derivative, BHAP U-90152).Other promising new non-nucleoside inhibitors (NNIs) that have beendeveloped to inhibit HIV RT include dihydroalkoxybenzyloxopyrimidine(DABO) derivatives, 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine(HEPT) derivatives, tetrahydrobenzondiazepine (TIBO),2′,5′-Bis-O-(tert-butyldimethylsilyl)-3′-spiro-5″-(4″-amino-1″,2″-oxathiole-2″,2′-dioxide)pyrimidine(TSAO), oxathiin carboxanilide derivatives, quinoxaline derivatives,thiadiazole derivatives, and phenetlhylthiazolylthioturea (PETT)derivatives.

[0013] NNIs have been found to bind to a specific allosteric site ofHIV-RT near tile polymerase site and interfere with reversetranscription by altering either the conformation or mobility of RT,thereby leading to a noncompetitive inhibition of the enzyme(Kohlstaedt, L. A. et al.,Science, 1992, 256, 1783-1790).

[0014] A number of crystal structures of RT complexed with NNIs havebeen reported (including α-APA, TIBO, Nevirapine, and HEPT derivatives),and such structural information provides the basis for furtherderivatization of NNI aimed at maximizing binding affinity to RT.However, the number of available crystal structures of RT NNI complexesis limited.

SUMMARY OF THE INVENTION

[0015] The invention provides substituted and unsubstitutedphenoxyethyl-thiourea-pyridine compounds which inhibit reversetranscriptase (RT) and which inhibit replication of a retrovirus, suchas human immunodeficiency virus-1 (HIV-1).

[0016] The invention additionally provides a method for inhibitingreverse transcriptase activity of a retrovirus, such as HIV-1,comprising contacting the retrovirus with a compound of the invention.The invention additionally provides a method for inhibiting replicationof a retrovitus, such as HIV-1, comprising contacting the retroviruswith a compound of the invention. The invention also provides a methodfor treating a retroviral infection in a subject, such as an HIV-1infection, comprising administering a compound of the invention to thesubject.

[0017] The invention also provides compositions comprising a compound orinhibitor of the invention, and optionally, an acceptable carrier. Inone embodiment, the composition is a pharmaceutical composition.

DETAILED DESCRIPTION OF THE INVENTION

[0018] Definitions

[0019] All scientific and technical terms used in this application havemeanings commonly used in the art unless otherwise specified. As used inthis application, the following words or phrases have the meaningsspecified.

[0020] As used herein, a “retrovirus” includes any virus that expressesreverse transcriptase. Examples of a retrovirus include, but are notlimited to, HIV-1, HIV-2, HTLV-I, HTLV-II, FeLV, FIV, SIV, AMV, MMTV,and MoMuLV.

[0021] As used herein, “reverse transcriptase (RT)” refers to an enzymehaving a non-nucleoside inhibitor (NNI) binding site similar to that ofHIV-1 RT and to which ligands which bind the composite binding pocket ofthe invention bind.

[0022] As used herein, “reverse transcriptase (RT) activity” means theability to effect reverse transcription of retroviral RNA to proviralDNA. One means by which RT activity can be determined is by measuringviral replication. One measure of viral replication is the p24 assaydescribed herein.

[0023] As used herein, a compound that “inhibits replication of humanimmunodeficiency virus (HIV)” means a compound that, when contacted withHIV-1, for example, via HIV-infected cells, effects a reduction in theamount of HIV-1 as compared with untreated control. Inhibition ofreplication of HIV-1 can be measured by various means known in the art,for example, the p24 assay disclosed herein.

[0024] As used herein, a “nonnucleoside inhibitor (NNI)” of HIV reversetranscriptase (HIV-RT) means a compound which binds to an allostericsite of HIV-RT, leading to noncompetitive inhibition of HIV-RT activity.Examples of nonnucleoside inhibitors of HIV-RT include, but are notlimited to, tetrahydroimidazobenzodiazepinthiones (TIBO),1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymines (HEPT),bis(heteroayl)piperazines (BHAP),2′-5′-bis-O-(tertbutyldimethylsilyl)-3′-spiro-5″-(4″-amino-1″,2″-oxathiole-2″, 2″-dioxide) pyrimidines (TSAO),dihydroalkoxybenzyloxopyrimidine (DABO) and phenethylthiazolylthiourea(PETT) analogs. The nonnucleoside inhibitor of HIV-RT of this inventionare substituted and unsubstituted phenoxyethyl-thiourea-pyridinecompounds

[0025] As used herein, “derivative” means a chemical substance derivablefrom a parent substance by addition or substitution of components andwhich maintains the activity of the parent substance.

[0026] As used herein, “halogen” includes fluoro, chloro, bromo andiodo.

[0027] As used herein, “pharmaceutically acceptable salt” refers to asalt that retains the desired biological activity of the parent compoundand does not impart any undesired toxicological effects. Examples ofsuch salts include, but are not limited to, (a) acid addition saltsformed with inorganic acids, for example hydrochloric acid, hydrobromicacid, sulfuric acid, phosphoric acid, nitric acid and the like; andsalts formed with organic acids such as, for example, acetic acid,oxalic acid, tartaric acid, succinic acid, maleic acid, furmaric acid,gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid,tannic acid, pamoic acid, alginic acid, polyglutamic acid,naphthalenesulfonic acids, naphthalenedisulfonic acids, polygalacturonicacid; (b) salts with polyvalent metal cations such as zinc, calcium,bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium,and the like; or (c) salts formed with an organic cation formed fromN,N′-dibenzylethylenediamine or ethylenediamine; or (d) combinations of(a) and (b) or (c), e.g., a zinc tannate salt; and the like. Thepreferred acid addition salts are the trifluoroacetate salt and theacetate salt.

[0028] As used herein, “pharmaceutically acceptable carrier” includesany material which, when combined with a compound of the invention,allows the compound to retain biological activity, such as the abilityto inhibit RT activity, and is non-reactive with the subject's immunesystem. Examples include, but are not limited to, any of the standardpharmaceutical carriers such as a phosphate buffered saline solution,water, emulsions such as oil/water emulsion, and various types ofwetting agents. Preferred diluents for aerosol or parenteraladministration are phosphate buffered saline or normal (0.9%) saline.Compositions comprising such carriers are formulated by well knownconventional methods (see, for example, Remington's PharmaceuticalSciences, Chapter 43, 14th Ed., Mack Publishing Col, Easton Pa. 18042,USA).

[0029] Compounds of the Invention

[0030] Compounds of the invention are phenoxyethyl-thiourea-pyridinecompounds that are useful as non-nucleoside inhibitors of reversetranscriptase. The compounds were designed as inhibitors of HIV-1 RT,based on a composite binding pocket computer model constructed from nine(9) individual crystal structures of RT-NNI complexes [51, 52]. Modelingstudies for rational drug design included the construction of acomposite NNI binding pocket for nine RT-NNI crystal structures, theanalyses of surface complementarity between NNIs and RT, and applicationof inhibitory constants (K_(i) values) combined with a docking procedureinvolving the novel thiourea compounds [51, 52]. This computationalapproach allowed the identification of several ligand derivatizationsites for the generation of more potent dual-function thioureacompounds. Detailed analysis of trovirdino-binding [51], revealedmultiple, specific sites which where larger functional groups could beincorporated in to the NNI. The composite binding pocket, the dockedtrovirdine molecule showed abundant sterically allowed usable spacesurrounding the pyridyl ring, the ethyl linker, and near the 5′-bromoposition.

[0031] The compounds of the invention are phenoxyethyl-thiourea-pyridinecompounds useful as non-nucleoside inhibitors of reverse transcriptasehaving the formula I:

[0032] The pyridine may be unsubstituted or substituted. For example R¹can be hydrogen, hydroxyl or halo.

[0033] Preferred compounds of the invention includeN-[2-(Phenoxy)ethyl]-N′-[2-(pyridyl)]thiourea (compound II),N-[2-(Phenoxy)ethyl]-N′-[2-(5-chloropyridyl)]thiourea (compound III),N-[2-(Phenoxy)ethyl]-N′-[2-(5-bromopyridyl)]thiourea (compound IV).

[0034] The compounds of the invention have the ability to inhibitreplication of a retrovirus, such as human immunodeficiency virus (HIV).In one embodiment, the compound inhibits replication of HIV with an IC₅₀of less than 50 μM, as determined by p24 enzyme immunoassay. In anotherembodiment, the compound inhibits replication of HIV with an IC₅₀ ofless than 5 μM. In another embodiment, the compound inhibits replicationof HIV with an IC₅₀ of less than 1 μM. In yet another embodiment, thecompound inhibits replication of HIV with an IC₅₀ of less than 5 nM. Inanother embodiment, the compound inhibits replication of HIV with anIC₅₀ of less than 1 nM.

[0035] The invention provides a composition comprising a compound orinhibitor of the invention, and optionally, an acceptable carrier. Inone embodiment, the composition is a pharmaceutical composition.Compositions of the invention are useful for prevention and treatment ofretroviral infection, such as HIV infection.

[0036] Methods of Using the Compounds of the Invention

[0037] The compounds of the invention are useful in methods forinhibiting reverse transcriptase activity of a retrovirus. Retroviralreverse transcriptase is inhibited by contacting RT in vitro or in vivo,with an effective inhibitory amount of a compound of the invention. Thecompounds of the invention also inhibit replication of retrovirus,particularly of HIV, such as HIV-1. Viral replication is inhibited, forexample, by contacting the virus with an effective inhibitory amount ofa compound of the invention.

[0038] The methods of the invention are useful for inhibiting reversetranscriptase and/or replication of multiple strains of HIV, includingmutant strains, and include treating a retroviral infection in asubject, such as an HIV-1 infection, by administering an effectiveinhibitory amount of a compound or a pharmaceutically acceptable acidaddition salt of a compound of the Formula I. The compound or inhibitorof Formula I is preferably administered in combination with apharmaceutically acceptable carrier, and may be combined with specificdelivery agents, including targeting antibodies and/or cytokines. Thecompound or inhibitor of the invention may be administered incombination with other antiviral agents, immunomodulators, antibioticsor vaccines.

[0039] The compounds of Formula I can be administered orally, parentally(including subcutaneous injection, intravenous, intramuscular,intrasternal or infusion techniques), by inhalation spray, topically, byabsorption through a mucous membrane, or rectally, in dosage unitformulations containing conventional non-toxic pharmaceuticallyacceptable carriers, adjuvants or vehicles. Pharmaceutical compositionsof the invention can be in the form of suspensions or tablets suitablefor oral administration, nasal sprays, creams, sterile injectablepreparations, such as sterile injectable aqueous or oleagenoussuspensions or suppositories. In one embodiment, the TET compounds ofthe invention can be applied intravaginally and/or topically, forexample in gel form, for prevention of heterosexual transmission of HIV.

[0040] For oral administration as a suspension, the compositions can beprepared according to techniques well-known in the art of pharmaceuticalformulation. The compositions can contain microcrystalline cellulose forimparting bulk, alginic acid or sodium alginate as a suspending agent,methylcellulose as a viscosity enhancer, and sweeteners or flavoringagents. As immediate release tablets, the conpositions can containmicrocrystalline cellulose, starch, magnesium stearate and lactose orother excipients, binders, extenders, disintegrants, diluents andlubricants known in the art.

[0041] For administration by inhalation or aerosol, the compositions canbe prepared according to techniques well-known in the art ofpharmaceutical formulation. The compositions can be prepared assolutions in saline, using benzyl alcohol or other suitablepreservatives, absorption promoters to enhance bioavailability,fluorocarbons or other solubilizing or dispersing agents known in theart.

[0042] For administration as injectable solutions or suspensions, thecompositions can be formulated according to techniques well-known in theart, using suitable dispersing or wetting and suspending agents, such assterile oils, including synthetic mono- or diglycerides, and fattyacids, including oleic acid.

[0043] For rectal administration as suppositories, the compositions canbe prepared by mixing with a suitable non-irritating excipient, such ascocoa butter, synthetic glyceride esters or polyethylene glycols, whichare solid at ambient temperatures, but liquefy or dissolve in the rectalcavity to release the drug.

[0044] Dosage levels of approximately 0.02 to approximately 10.0 gramsof a compound of the invention per day are useful in the treatment orprevention of retroviral infection, such as HIV infection, AIDS orAIDS-related complex (ARC), with oral doses 2 to 5 times higher. Forexample, HIV infection can be treated by administration of from about0.1 to about 100 milligrams of compound per kilogram of body weight fromone to four times per day. In one embodiment, dosages of about 100 toabout 400 milligrams of compound are administered orally every six hoursto a subject. The specific dosage level and frequency for any particularsubject will be varied and will depend upon a variety of factors,including the activity of the specific compound the metabolic stabilityand length of action of that compound, the age, body weight, generalhealth, sex, and diet of the subject, mode of administration, rate ofexcretion, drug combination, and severity of the particular condition.

[0045] The compounds of Formula I can be administered in combinationwith other agents useful in the treatment of HIV infection, AIDS or ARC.For example, the compound of the invention can be administered incombination with effective amounts of an antiviral, immunomodulator,anti-infective, or vaccine. The compound of the invention can beadministered prior to, during, or after a period of actual or potentialexposure to retrovirus, such as HIV.

[0046] Conjugation to a Targeting Moiety

[0047] The compound of the invention can be targeted for specificdelivery to the cells to be treated by conjugation of the compounds to atargeting moiety. Targeting moiety useful for conjugation to thecompounds of the invention include antibodies, cytokines, and receptorligands expressed on the cells to be treated.

[0048] The term “conjugate” means a complex formed with two or morecompounds.

[0049] The phrase “targeting moiety” means a compound which serves todeliver the compound of the invention to a specific site for the desiredactivity. Targeting moieties include, for example, molecules whichspecifically bind molecules present on a cell surface. Such targetingmoieties useful in the invention include anti-cell surface antigenantibodies. Cytokines, including interleukins, factors such as epidermalgrowth factor (EGF), and the like, are also specific targeting moietiesknown to bind cells expressing high levels of their receptors.

[0050] Particularly useful targeting moieties for targeting thecompounds of the invention to cells for therapeutic activity includethose ligands that bind antigens or receptors present on virus-infectedcells to be treated. For example, antigens present on T-cells, such asCD48, can be targeted with antibodies. Antibody fragments, includingsingle chain fragments, can also be used. Other such ligand-receptorbinding pairs are known in the scientific literature for targetinganti-viral treatments to target cells. Methods for producing conjugatesof the compounds of the invention and the targeting moieties are known.

[0051] Methods of Making the Compounds of the Invention

[0052] The compounds of the invention are prepared as follows. First anamine substituted pyridine is reacted with thiocarbonyldiimidazole inacetonitrile at room temperature. The reaction precipitate is thenfiltered to yield the thiocarbonlyl intermediate. Dimethyl formamide wasthen added to the thiocarbonyl intermediate to create a solution. Anappropriate amine was then added to the thiocarbonlyl intermediatesolution, and the resulting solution was heated. The solution wascooled, precipitated, filtered, and the solvent evaporated to yield thedesired thiourea compound. The compounds were further purified by columnchromatography. The compounds of the invention are prepared as depictedin Scheme I below:

EXAMPLES

[0053] The following examples are presented to illustrate the presentinvention and to assist one of ordinary skill in making and using thesame. The examples are not intended in any way to otherwise limit thescope of the invention.

Example 1

[0054] Synthesis and Characterization of Compounds of the Invention

[0055] All chemicals were purchased from the Aldrich Chemical Company,Milwaukee, Wis., and were used directly for synthesis. Anhydroussolvents such as acetonitrile, methanol, ethanol, ethyl acetate,tetrahydrofuran, chloroform, and methylene chloride were obtained fromAldrich as sure seal bottles under nitrogen and were transferred toreaction vessels by cannulation. All reactions were carried out undernitrogen atmosphere.

[0056] Synthesis of Compounds

[0057] The general synthesis from Scheme I above was followed. Thespecific amine substituted pyridines utilized to make Compounds I, II,and III respectively were 2-aminopyridine, 2-amino-5-chloropyridine, and2-amino-5-bromopyridine.

[0058] Specifically, thiocarbonyldiimidazole (8.90 g, 50 mmol) and theamino substituted pyridine (50 mmol) were added to 50 mL of dryacetonitrile at room temperature. The reaction mixture was stirred for12 hours and the precipitate filtered, washed with cold acetonitrile(2×25 mL), and dried under vacuum to afford (1 1.40 g, 80% ) of compoundA. To a suspension of compound A (0.55 eqv) in dimethyl formamide (15mL) an appropriate amine (0.50 eqv) was added. The reaction mixture washeated to 100° C. and stiffed for 15 hours. The reaction mixture waspoured into ice-cold water and the suspension was stirred for 30minutes. The product was filtered, washed with water, dried, and furtherpurified by column chromatography to furnish the target compounds ingood yields. Trovidine, a comparative standard, was prepared by themethod described in Bell et al.,J. Med. Chem 1995,38:4926-9; Ahgrenet.al., 1995, Antimicrob.Agents Chemotheraphy 39:1329-1335.

[0059] Characterization of Compounds

[0060] Proton and carbon nuclear magnetic resonance spectra wererecorded on a Varian spectrometer using an automatic broad band probe.Unless otherwise noted, all NMR spectra were recorded in CDCl₃ at roomtemperature. The chemical shifts reported are in parts per millionrelative to tetramethyl silane as standard. The multiplicity of thesignals were designated as follows: s, d, dd, t, q, m which correspondsto singlet, doublet, doublet of doublet, triplet, quartet and multipletrespectively. UV spectra were recorded from a Beckmann Model #DU 7400UV/V is spectrometer using a cell path length of 1 cm. Fourier TransformInfrared spectra were recorded using an FT-Nicolet model Protege #460instrument. The infrared spectra of the liquid samples were run as neatliquids using KBr discs. Mass spectrum analysis was conducted usingeither a Finnigan MAT 95 instrument or a Hewlett-Packard Matrix AssistedLaser Desorption (MALDI) spectrometer model #G2025A. The matrix used inthe latter case was cyano hydoxy cinnamic acid. Melting points weredetermined using a Melt John's apparatus and uncorrected. Elementalanalysis was performed by Atlantic Microlabs (Norcross, Ga.). Columnchromatography was performed using silica gel obtained from the BakerCompany. The solvents used for elution varied depending on the compoundand included one of the following: ethyl acetate, methanol, chloroform,hexane, methylene chloride and ether. Characterization data for thesynthesized compounds is shown below:

[0061] N-[2-(Phenoxy)ethyl]-N′-[2-(pyridyl)thiourea (Compound II):

[0062] Yield 60%; mp. 168.5-170.5° C.; UV(MeOH) λ_(max)224, 246, 267,293 nm; IR ν, 3232, 3045, 2931, 1602, 1560, 1481, 1317, 1245, 1080, 773,668 cm⁻¹; ¹H NMR (DMSO) δ11.98 (t, 1 H), 10.67 (s, 1 H), 8.17 (dd. 1 H,J=5.4), 7.88-7.73 (m, 1 H), 7.31-7.17 (m, 2 H), 7.15 (dd. 1 H, J=8.4),7.04-6.90 (m, 4 H), 4.19 (t, 2 H). 4.00 (q, 2 H); ¹³C NMR(DMSO-d₆)δ179.8. 158.2, 153.7, 145.5, 139.0, 129.6, 120.9, 118.0, 114.6, 112.6,65.7, 44.0; MALDI-TOF: 275.0.

[0063] N-[2-(Phenoxy)ethyl]-N′-[2-(5-chloropyridyl)thiourea. (CompoundIII):

[0064] Yield 65%; mp. 168-169° C.; UV (MeOH) λ_(max): 245, 265 nm; IR ν3161, 3088, 3037, 2933, 2875, 1601, 1562, 1533, 1477, 1407, 1359, 1305,1263, 1238, 1194, 1136, 1111, 1047, 908, 862, 821, 750, 690 cm⁻¹; ¹H NMR(DMSO-d₆) δ11.50 (t, 1 H), 10.83 (s, 1 H), 8.19 (d, 1 H, J=2.7), 7.86(dd, 1 H, J=8.7), 7.28 (q, 2 H), 7.20 (d, 1 H, J=9), 7.00-6.90 (m, 3 H),4.19 (t, 2 H), 3.99 (q, 2 H);¹³C NMR (DMSO-d₆) δ179.6, 158.2, 152.1,143.8, 138.9, 129.6, 123.9, 120.9, 114.6, 114.2, 65.6, 44.1.

[0065] N-[2-(Phenoxy)ethyl]-N′-[2-(5-bromopyridyl)]thiourea (CompoundIV):

[0066] Yield 56%; mp.162-163° C.; UV (MeOH) λ_(max): 249, 268 nm; IR ν3219, 3161 3084, 3032, 2929, 2875, 1599, 1560, 1527, 1468 1356, 1307,1278, 1238, 1191, 1138, 1078,1047, 1005, 951, 860, 821, 752, 690 cm⁻¹;¹H NMR (DMSO-d₆) δ11.48 (t, 1 H), 10.82 (s, 1 H), 8.26 (d, 1 H, J=2.4),7.97 (dd, 1 H, J=9), 7.27 (q, 2 H), 7.14 (dd, 1 H, J=8.7), 7.00-6.90 (m,3 H), 4.18 (t, 2 H), 3.99 (q, 2 H;¹³C NMR (DMSO-d₆) δ179.6, 158.2,152.3, 146.0, 141.5, 129.6, 120.9, 114.6, 112.1, 65.6, 44.2.

Example 2

[0067] In vitro Inhibition of Reverse Transcriptase by the Compounds ofthe Invention

[0068] The synthesized compounds were tested for RT inhibitory activity(IC₅₀[rRT]) against purified recombinant HIV RT using the cell-freeQuan-T-RT system (Amersham, Arlington Heights, Ill.), which utilizes thescintillation proximity assay principle as described in Bosworth, etal., 1989, Nature 341:167-68. In the assay, a DNA/RNA template is boundto SPA beads via a biotin/strepavidin linkage. The primer DNA is a16-mer oligo(T) which has been annealed to a poly(A) template. Theprimer/template is bound to a strepavidin-coated SPA bead.

[0069]³H-TTP is incorporated into the primer by reverse transcription.In brief,³H-TTP, at a final concentration of 0.5 μCi/sample, was dilutedin RT assay buffer (49.5 mM Tris-Cl, pH 8.0, 80 mM KCl, 10 Mm MgCl₂, 10mM DTT, 2.5 mM EGTA, 0.05% Nonidet-P-40), and added to annealed DNA/RNAbound to SPA beads. The compound being tested was added to the reactionmixture at 0.001 μM-100 μM concentrations. Addition of 10 mU ofrecombinant HIV RT and incubation at 37° C. for 1 hour resulted in theextension of the primer by incorporation of H-TTP. The reaction wasstopped by addition of 0.2 ml of 120 mM EDTA. The samples were countedin an open window using a Beckman LS 7600 instrument and IC₅₀ valueswere calculated by comparing the measurements to untreated samples.

[0070] In addition, the anti-HIV activity of the compounds was measuredby determining their ability to inhibit the replication of the HIV-1strain HTLVIIIB in peripheral blood mononuclear cells (PBMC) fromhealthy volunteer donors, using the method described in Uckun et.al.,1998, Antimicrobial Agents and Chemotheraphy 42:383. The HIV strainHTLVIIIB was kindly provided by Dr. Neal T. Wetherall, VIROMEDLaboratories, Inc., and was propagated in CCRF-CEM cells.

[0071] The data are shown in Table 1A. Compounds II, III, and IV eachexhibited promising anti-HIV activity with 100% inhibition atconcentrations≧1 μM, minimal cytotoxicity with CC₅₀ values ranging from30 to >100 μM, and selectivity indices ranging from 1100 to >50,000.Compared with phenethyl thiourea pyridine compounds (shown in Table 1Bbelow) the phenoxyethyl thiourea pyridine compounds have greaterpotency. Taken together, these results provide evidence that thestructural features of the “bridge” between the pyridyl and phenylmoieties of PETT-related thiourea compounds significantly affects theirbiologic activity as NNI of HIV-1 RT. TABLE 1A HIV-RT inhibitoryactivity of the compounds of the invention Compound IC₅₀[rRT]IC₅₀[HTLV_(IIIB)] CC Number Structure μM μm μM SI II

10.1 0.005 55 1,100 III

 1.5 0.004 >100 >25,000 IV

 1.5 0.005 92 18,400 Troviridine

ND 0.007 100 14,286

[0072] TABLE 1B HIV-RT inhibitory activity of phenethyl thioureapyridine compounds. Compound IC₅₀ [rRT] IC₅₀[HTLV_(III8)] CC NumberStructure μM μm μM SI V

2.5 20.8 N.D. N.D. VII

87.7 3.067 >100 N.D. Troviridine

0.8 0.007 >100 N.D.

[0073] All publications, patents, and patent documents described hereinare incorporated by reference as if fully set forth. The inventiondescribed herein may be modified to include alternative embodiments. Allsuch obvious alternatives are within the spirit and scope of theinvention, as claimed below.

We claim:
 1. A compound of the formula:

wherein R¹ is H or an electron withdrawing group; n is an integer from 0to 5; R is hydrogen, C₁-C₆ alkyl, alkoxy, hydroxy, amino, or halo.
 2. Acompound of the formula:

wherein R¹ is cyclo(C₃-C₁₂) alkyl, cyclo(C₃-C₁₂) alkenyl, isothiazolyl,tetrazolyl, triazolyl, pyridyl, imidazolyl, phenyl, napthyl,benzoxazolyl, benzimidazolyl, thiazolyl, oxazolyl, benzothiazolyl,pyrazinyl, pyridazinyl, thiadiazolyl, benzotriazolyl, pyrolyl, indolyl,benzothienyl, thienyl, benzofuryl, quinolyl, isoquinolyl, or pyrazolyl;n is an integer from 0 to 5; R is hydrogen, C₁-C₆ alkyl, alkoxy,hydroxy, amino, or halo.
 3. A compound of claim 1 wherein R₁ is hydrogenor a halo; and n is 0
 4. A compound of the formula:


5. A compound of the formula:


6. A compound of the formula:


7. A pharmaceutical composition comprising a therapeutically effectiveamount of the compound of claim 1 and a pharmaceutically acceptablecarrier or diluent.
 8. A pharmaceutical composition comprising atherapeutically effective amount of the compound of claim 2 and apharmaceutically acceptable carrier or diluent.
 9. A pharmaceuticalcomposition comprising a therapeutically effective amount of thecompound of claim 3 and a pharmaceutically acceptable carrier ordiluent.
 10. A pharmaceutical composition comprising a therapeuticallyeffective amount of the compound of claim 4 and a pharmaceuticallyacceptable carrier or diluent.
 11. A method for inhibiting HIV reversetranscriptase comprising contacting said HIV with an effectiveinhibitory amount of a compound selected from claims 1, 2, 3, or
 4. 12.A method for treating HIV infection in a subject comprisingadministering to said subject an anti-HIV effective amount of a compoundselected from claims 1, 2, 3, or
 4. 13. A method for treatingtherapy-naive or drug-resistant HIV in a subject comprisingadministering to said subject an effective amount of at least onecompound of claims 1, 2, 3, or
 4. 14. A medicament for treatment of apatient with HIV infection comprising at least one compound of claims 1,2, 3, or 4; and a pharmaceutically acceptable carrier.
 15. A medicamentfor treatment of a patient with AIDS comprising at least one compound ofclaims 1, 2, 3, or 4; and a pharmaceutically acceptable carrier.
 16. Amedicament for inhibiting HIV reverse transcriptase comprising at leastone compound of claims 1, 2, 3, or 4; and a pharmaceutically acceptablecarrier.